The complexity and multifactorial nature of Alzheimer?s disease (AD) pose unique challenges for mechanistic studies and developing therapies. Although many transgenic mouse models have been generated for AD research and these models are important for our understanding of the pathological basis of the disease, none of them has captured the entire spectrum of the disease pathologies. This is likely due to significant species differences between mouse and human neural cells. Therefore, there is an urgent need to establish human disease modeling platforms to complement studies in animal models for AD research. Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, human iPSCs (hiPSCs) have been widely used for disease modeling and drug discovery. However, given the relative immaturity of cells differentiated from hiPSCs, it is challenging to use them for modeling late-onset diseases, such as AD, for which cellular aging is important in disease pathologies. Direct reprogramming is an alternative cellular reprogramming technology, which allows direct conversion of one type of cells, such as fibroblasts, into another type of cells, such as neural stem cells (NSCs) or neurons. It has been shown that direct reprogramming enables generation of human neurons that possess key elements of cellular aging, because this reprogramming process does not go through the iPSC stage involving extensive epigenetic modifications. While the strongest risk factor for AD is aging, the strongest genetic risk factor of AD is apolipoprotein E4 (apoE4). Among the three isoforms of human apoE (apoE2, apoE3, and apoE4), apoE4 increases AD risk, apoE3 is neutral, and apoE2 is protective. Although the roles of apoE4 in AD pathogenesis have been extensively studied, the protective roles of apoE2 in AD have been surprisingly understudied. Clearly, better understanding of the molecular and cellular mechanisms underlying apoE2?s protective roles in AD will likely provide novel targets or signaling pathways for anti-AD drug development, especially for late-onset AD. The objectives of this proposal are to develop aging-relevant human neuron models of late-onset AD, using direct reprogramming technology in combination with CRISPR/Cas9-mediated gene editing, and to dissect the underlying mechanisms of apoE2?s protective roles in AD. We propose three complementary aims to accomplish the goals. Aim 1: To generate isogenic human fibroblast lines with an apoE2/2 or apoE3/3 genotype from the parental human fibroblast lines with an apoE4/4 genotype as cell sources for direct reprogramming. Aim 2: To dissect the protective roles of apoE2 and their underlying mechanisms using directly reprogrammed human neurons and astrocytes. Aim 3: To dissect the protective roles of apoE2 and their underlying mechanisms using directly reprogrammed NSC-derived neurons and astrocytes. The outcomes of the proposed studies will promote our understanding of the molecular and cellular mechanisms underlying apoE2?s protective roles in AD, and will likely identify novel targets for anti-AD drug development.